This method allows to elucidate the microscale ecology and behavior of microorganisms navigating dynamic chemical gradients and to uncover their hidden trade-offs, nutrient kinetics, and population dynamics in ecologically relevant microenvironments. By combining microfluidic technology with photolysis, we generate controlled chemical pulses, where localized chemoattractant becomes suddenly available at the microscale, to measure the chemotactic response of microbial populations first exposed to unsteady chemical gradients. Design the channel using CAD software, and print it onto a transparency film to create the photo mask.
Fabricate the master by soft lithography in a clean room, according to the manuscript. Prepare a PDMS mixture by combining the elastomer with its curing agent at a 10-to-one ratio in a 40-milliliter beaker. With a plastic knife, mix vigorously until the liquid is homogeneous.
Immediately degas the PDMS mixture in a vacuum chamber for 45 minutes at room temperature. To expedite the process, periodically release the vacuum in order to burst the bubbles that form at the interface. Remove dust from the surface of the master with a pressurized cleaner.
Then pour the degassed PDMS mixture onto the master, and bake it in an oven at 80 degrees Celsius for two hours. Cut the PDMS with a blade around the microstructures at a distance of approximately five millimeters, and then carefully peel the PDMS from the master. Punch holes to serve as the inlet and outlet of the microchannel.
Seal the microchannel with adhesive tape to prevent accumulation of dust. Design the 3D shape using 3D design software, and print the master for the PDMS mold with a high-resolution 3D printer. After preparing and degassing the PDMS mixture as done previously, clean the surface of the master by dabbing with adhesive tape.
Put the master on a scale. Then, while avoiding the central region of the master, pour 23.4 grams of uncured PDMS mixture in order to obtain the desired height of PDMS by matching the height of the master at 0.5 millimeters. Remove any remaining bubbles with the help of compressed air.
Place the PDMS cast on the master in an oven at 45 degrees Celsius to bake for at least 12 hours. Then, gently peel off the hardened PDMS layer, and punch inlet and outlet holes for the injection of the bacterial suspension. Activate both the surface of a Petri dish and the PDMS mold with oxygen plasma for two minutes.
Bond the PDMS mold on the Petri dish by gently pressing the mold to the Petri dish. Avoid pressing where the features are located. Place the Petri dish bonded to the PDMS 3D mold in an oven at 45 degrees Celsius for at least 12 hours to strengthen the chemical bond.
Next, activate the nanoporous polycarbonate membrane in an oxygen plasma chamber for one minute at room temperature. Under a chemical hood, dilute a commercial solution of 3-aminopropyltriethoxysilane in deionized water to 1%by volume by using a polypropylene tube. Transfer the diluted 3-aminopropyltriethoxysilane solution in a Petri dish, and immerse the activated membrane in the 3-aminopropyltriethoxysilane solution for 20 minutes.
Then, remove the membrane from the 3-aminopropyltriethoxysilane solution with tweezers, and place it on a clean room wipe to dry. For the fabrication of the PDMS microfluidic channels that will lie on the membrane and allow washing of the bacterial arena, repeat the fabrication of the microfluidic device for the single chemical pulse experiment as done previously. To bond the PDMS washing channels to the functionalized membrane, first activate both the PDMS washing channel and the polycarbonate membrane with an oxygen plasma chamber for two minutes.
Immediately after the plasma treatment, bring the functionalized membrane and the PDMS washing channel into contact by gently pressing the PDMS washing channel onto the membrane. It is important to gently press together the PDMS and the membrane, or the membrane might be irreversibly bonded to the ceiling of the 200-micrometer height PDMS microchannel. Then, activate both the bonded laminate PDMS washing channel polycarbonate membrane and the 3D PDMS mold previously bonded to the Petri dish by placing them into an oxygen plasma chamber for two minutes.
Sandwich the membrane between two PDMS layers, and press together. Place the Petri dish bonded to the sandwich structure in an oven at 45 degrees Celsius for at least 12 hours to strengthen the chemical bond. To begin, maintain the millifluidic chamber under vacuum for 20 minutes.
Then, place the Petri dish containing the millifluidic chamber onto a microscope stage. With a pipette, fill the chamber below the membrane with the dilute bacterial suspension of GFP-fluorescent Vibrio ordalii in one-millimolar 4-methoxy-7-nitroindolinyl-caged-L-glutamate solution in artificial seawater. After filling the channel with the bacterial suspension, suck the solution in excess with a paper towel, and seal inlet and outlet with PDMS plugs by gently pressing them into the holes.
Fill a syringe with an artificial seawater solution of one-millimolar 4-methoxy-7-nitroindolinyl-caged-L-glutamate, and attach tubing to the inlet and outlet of the washing channel above the membrane. Connect the tubing to a waste reservoir, and ensure that the tubing is entirely submerged in the fluid waste reservoir to avoid pressure oscillations. Set the flow rate on the syringe pump to 50 microliters per minute.
Start the syringe pump to establish the flow in the washing channel above the membrane. Start the software controlling the LED beam and the microscope stage, and generate user-defined sequences of pulses at different locations and with different intensities. Record video using a 4x objective at regular time intervals over a period of several hours and over multiple contiguous locations to cover a large surface.
In this study, the microfluidic and millifluidic devices were used to study bacterial chemotactic response and accumulation profiles under dynamic nutrient conditions. Maximum intensity projections show the bacterial position, indicated by white traces, over a 0.5-second interval immediately following the pulse release and 40 seconds after the pulse release. The bacterial trajectories were also shown in black 60 seconds after the pulse release.
The shaded region represents the chemical pulse released by photolysis at time zero second in the middle of the field of view, which subsequently diffused. The bacterial concentration, as well as the temporal dynamics of the radial drift velocity following a pulse release in the center of the field of view are shown here. Negative values of the drift velocity correspond to directed chemotactic motion towards the center of the pulse.
Swimming statistics are presented here for a bacterial population in the absence of chemical gradients. Trajectories represent the two-dimensional projections of the three-dimensional bacterial motion in the microchannel. Probability distribution of measured bacterial swimming speed was fit by a gamma distribution.
From the bacterial trajectories, the probability distribution of the time between successive reorientations was extracted. The reorientation pattern, the distribution of swimming speed, and the reorientation statistics of the organisms were used to inform an individual based model. This method has been tested on the marine bacteria Vibrio ordalii performing chemotaxis toward the amino acid glutamate, but the proposed methodology is broadly applicable to different combination of species and chemoattractants, as well as to biological processes beyond chemotaxis, such as population and community dynamics in spatially heterogeneous and dynamic conditions.
Classical techniques in microbial ecology and microbiology, such as growths in batch cultures or chemostats, have largely ignored the spatio-temporal characteristics of microbial habitats. The near-instantaneous creation of chemical pulses at the microscales by photolysis aims to reproduce the type of nutrient pulses that marine bacteria encounter in the wild from a range of sources, for example, the diffusive spreading of plumes behind sinking organic particles or the nutrient spreading from lysed phytoplankton cells. The aminosilane solution is acutely toxic and should be handled with extreme care.
We work exclusively under the fume hood and wear protective equipment like lab coat, safety goggles, and gloves. We also take care of the waste we generated.
